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SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies., This work was supported by grants CV20.26565 from the Consejería de Economía y Conocimiento, Junta de Andalucía (Spain), PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033), and co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future”. The work performed in C.M.'s laboratory was supported by grants from ANRS (Agence Nationale de Recherches sur le SIDA et les hépatites virales), the Investissements d'Avenir program managed by the ANR under reference ANR-10-LABX-77 and EHVA (No. 681032, Horizon 2020). Work in S.B.'s laboratory was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR-11-LABX-0070_TRANSPLANTEX), the INSERM (UMR_S 1109), the Institut Universitaire de France (IUF), all the University of Strasbourg (IDEX UNISTRA), the European Regional Development Fund (European Union) INTERREG V program (project no. 3.2 TRIDIAG) and MSD-Avenir grant AUTOGEN. We are grateful to the Spanish Radiation Synchrotron Source (ALBA), Barcelona, Spain and the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and staff assistance at XALOC (ALBA) and ID30B and ID23-2 (ESRF) beamlines during diffraction data collection. We thank María Carmen Salinas-García for her assistance in carrying out the crystallization screenings. We also thank Pilar González-García for helping us with the statistical analysis.

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene- 1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP., Spanish State Research Agency, SRA/ 10.13039/501100011033 (grants BIO2016-76640-R and PID2019.107515RB.C21), ERDF/ESF

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region., Spanish Ministry of Economy and Competitiveness (grant: BIO2016-76640-R), ANRS and the Vaccine Research Institute for the Investissements d'Avenir program to C.M. and by the European Fund for Research and Development from the European Union., Departamento de Química Física, Facultad de Ciencias, Universidad de Granada. Grupo FQM-171 "Biofísica y Biotecnología Molecular"

Pentacyclic triterpenes, such as oleanolic acid (I), are promising scaffolds for diversification through the use of combinatorial methods to obtain derivatives that improve their biological properties, increasing their bioavailability and enhancing their therapeutic efficacy. The purpose of this study was to evaluate the influence that derivatives of oleanolic acid, conjugated with one or two amino acids and an acyl group, might exert on HIV-1 protease inhibition. The in vitro studies conducted suggested that the presence of a carboxyacyl group generally improves the inhibition of HIV-1 protease, especially when a phthaloyl group is present, with IC50 concentration values below 5 μM. The gain in activity of three 3-phthaloyl derivatives, with sub-micromolar IC50 values, was between 60- and 100-fold more active than oleanolic acid. A molecular docking study has also been performed to elucidate the mode of binding to the protease by these oleanolic acid derivatives. In general, the derivatives that exhibited the highest inhibitory activity of HIV-1 protease also showed the highest binding energies in docking simulations. The overall results suggest that the coupling of one or two amino acids and a phthaloyl group to oleanolic acid improves HIV-1 protease inhibition, implying that these triterpene derivatives may be promising antiviral agents against HIV.

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR–NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches., Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain, Ministerio de Economía y Competitividad (grants BIO2016-76640-R and BIO2016-78020-R) y Fondo Europeo para el Desarrollo Regional (FEDER) de la Unión Europea, ANRS and the Vaccine Research Institute for the Investissements d'Avenir program managed by the ANR under reference ANR-10-LABX-77

The functional and pathological implications of the enormous genetic diversity of the human genome are mostly unknown, primarily due to our unability to predict pathogenicity in a high-throughput manner. In this work, we characterized the phenotypic consequences of eight naturally-occurring missense variants on the multifunctional and disease-associated NQO1 protein using biophysical and structural analyses on several protein traits. Mutations found in both exome-sequencing initiatives and in cancer cell lines cause mild to catastrophic e ects on NQO1 stability and function. Importantly, some mutations perturb functional features located structurally far from the mutated site. These e ects are well rationalized by considering the nature of the mutation, its location in protein structure and the local stability of its environment. Using a set of 22 experimentally characterized mutations in NQO1, we generated experimental scores for pathogenicity that correlate reasonably well with bioinformatic scores derived from a set of commonly used algorithms, although the latter fail to semiquantitatively predict the phenotypic alterations caused by a significant fraction of mutations individually. These results provide insight into the propagation of mutational e ects on multifunctional proteins, the implementation of in silico approaches for establishing genotype-phenotype correlations and the molecular determinants underlying loss-of-function in genetic diseases., ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00 SAF2015-69796, Junta de Andalucía P18-RT-2413

HIV-1 glycoprotein 41 (gp41) mediates fusion between virus and target cells by folding into a fusion active state, in which the C-terminal heptad repeat (CHR) regions associate externally to the N-terminal heptad repeat (NHR) trimer and form a very stable six-helix bundle coiled-coil structure. Therefore, interfering with the NHR-CHR interaction of gp41 is a promising therapeutic approach against HIV-1. However, a full understanding of the molecular and mechanistic details of this interaction is still incomplete. Here, we use single-chain, chimeric proteins (named covNHR) that reproduce accurately the CHR-NHR interactions to analyze the binding thermodynamics of several peptides with different length from the CHR region. The results indicate that cooperative binding involving two or more pockets of the NHR groove is necessary to obtain relevant affinities and that the binding energy is broadly distributed along the interface, underlining a crucial role of a middle pocket to achieve tight binding. In contrast, targeting only the deep hydrophobic pocket is insufficient to achieve significant affinity. Moreover, calorimetry experiments in combination with limited proteolysis performed using a mutant with occluded binding in the N-terminal pocket reveal the existence of an allosteric communication between the different regions. This study is the first detailed thermodynamic dissection of the NHR-CHR interaction in gp41 and contributes therefore to a better understanding of HIV fusion. These results are relevant for the development of potential fusion inhibitors., Grant BIO2016-76640-R from the Spanish Ministry of Economy and Competitiveness and by the European Fund for Research and Development from the European Union., Departamento de Química Física, Facultad de Ciencias, Universidad de Granada. Grupo FQM-171 "Biofísica y Biotecnología Molecular"